Use of urea variants as affinity ligands

ABSTRACT

The present invention relates to an IgG-binding compound, which more specifically has affinity for human IgGs of κ-type and functional derivatives thereof. More specifically, the compound according to the invention comprises an N,N-alkylated urea moiety located between an aromatic part and another part, which is a linear or cyclic substituted or unsubstituted aliphatic group. The compound binds to a pocket-shaped binding site present on all human IgG κ-Fabs, which site is located between the two domains (CH1 and CL) of its constant part. Accordingly, the compound according to the invention is a ligand for human IgGs of κ-type, and consequently, the invention also relates to a separation matrix for affinity chromatography, which matrix comprises said compound, as well as to other uses of the compound.

TECHNICAL FIELD

The present invention relates to a novel IgG-binding compound useful asa ligand for human IgGs of κ-type and functional derivatives thereof.The invention also relates to a separation matrix for use in affinitychromatography comprising said compound and various uses thereof.

BACKGROUND

Antibodies, also denoted immunoglobulins, are normally synthesised bylymphoid cells derived from B-lymphocytes of bone marrow. Lymphocytesderived from the same clone produce immunoglobulin of a single aminoacid sequence. Lymphocytes cannot be directly cultured over long periodsof time to produce substantial amounts of their specific antibody.However, a process of somatic cell fusion, specifically between alymphocyte and a myeloma cell, has been shown to yield hybrid cells thatgrow in culture and produce a specific antibody known as a monoclonalantibody. The resulting hybrid cell is known as a hybridoma. Amonoclonal antibody belongs to a group of antibodies whose population issubstantially homogeneous, i.e. the individual molecules of the antibodypopulation are identical except for naturally occurring mutations.

The development of monoclonal antibody technology has provided anenormous opportunity for science and medicine in implementing research,diagnosis and therapy. Monoclonal antibodies are e.g. used inradioimmunoassays, enzyme-linked immunosorbent assays,immunocytopathology, and flow cytometry for in vitro diagnosis, and invivo for diagnosis and immunotherapy of human disease.

Antibodies are grouped into five different types, namely immunoglobulinG (IgG), which is the most prevalent; immunoglobulin A (IgA);immunoglobulin M (IgM); immunoglobulin D (IgD); and immunoglobulin E(IgE). At present, about thirty percent of the biotechnology-deriveddrugs under development are based on monoclonal antibodies of type G.

The Y-shaped disposition of the structure of the IgG molecule is wellknown from standard biochemistry textbooks. In brief, regarding itstertiary structure, one intact IgG molecule consists of six globularregions, each of which is formed by two domains. Regarding its primarystructure, an IgG consists of two light chains and two heavy chains,which are covalently linked by disulphide bridges. The two globularparts that correspond to the “base of the Y” form the Fc fragment andare formed by domains consisting of only heavy chain residues. Contraryto this, each of the “arms of the Y” constitutes a Fab fragment with twoglobular parts each. Each of the globular parts in a Fab fragment isformed when one domain from the light chain contacts one domain from theheavy chain. It is well known that the globular part located furtheraway from the centre of the antibody comprises the regions known as thehypervariable regions as well as the antigen-binding site.

By sequence homology, heavy chains of IgGs can be classified into thefour types 1, 2, 3 and 4 whereas light chains fall into two types calledλ and κ. In humans, about 40% of the IgG molecules carry a light chainof λ type whereas about 60% carry a light chain of κ type. IgGs built upof both light and heavy chains inherit both types of partitionings.Accordingly, one partitioning divides IgGs into four subclasses IgG1,IgG2, IgG3 and IgG4 as compared to the second partitioning which dividesIgGs into two subtypes λ and κ. The same type of classification can beapplied to antibody fragments like Fab fragments and so called F(ab′)₂fragments, which consist of two Fab fragments connected by a disulphide.

These days, IgGs are generated according to standard techniques in largequantities in cellular expression systems. The most widely usedproduction method includes purification via chromatography, which due toits versatility and sensitivity to the compounds often is the preferredpurification method in the context of biomolecules. The termchromatography embraces a family of closely related separation methods,which are all based on the principle that two mutually immiscible phasesare brought into contact. More specifically, the target compound isintroduced into a mobile phase, which is contacted with a stationaryphase. The target compound will then undergo a series of interactionsbetween the stationary and mobile phases as it is being carried throughthe system by the mobile phase. The interactions exploit differences inthe physical or chemical properties of the components in the sample. Theinteractions can be based on one or more different principles, such ascharge, hydrophobicity, affinity etc. In the context of antibodies,affinity chromatography is the most widely utilised purification scheme.More specifically, affinity chromatography is a highly specific mode ofchromatography wherein molecular recognition process takes place betweena biospecific ligand and a target substance by a principle of lock-keyrecognition, which is similar to the enzyme binding to a receptor. For ageneral review of the principles of affinity chromatography, see e.g.Wilchek, M., and Chaiken, I. 2000. An overview of affinitychromatography. Methods Mol. Biol. 147: 1-6.

Lawrence et al (J. F. Lawrence, C. Ménard, M-C Hennion, V. Pichon, F. LeGoffic, N. Durand in Journal of Chromatography A, 732 (1996) 277-281:Use of immunoaffinity chromatography as a simplified cleanup techniquefor the liquid chromatographic determination of phenylurea herbicides inplant material) describes an evaluation of polyclonal antibodies forcleanup of extracts of food samples. More specifically, antibodies weregenerated in rabbit after inoculations with an antigen prepared from anurea herbicide. Thus, the antibodies were highly specific to the ureaherbicide, which is consequently not useful in any method of generalantibody purification.

Another application of urea compounds is provided in EP 0 743 067 (TorayIndustries), wherein the compounds are presented as highly selectiveadsorbing materials used for elimination or detoxification ofsuperantigens from body fluids. The superantigens described areenterotoxins and exotoxins, which are large proteins.

In the field of affinity chromatography, various patents and patentapplications relate to protein A, which is an IgG-binding cell wallprotein of the bacteria Staphylococcus aureus, and its use as a ligand.For example, PCT/SE83/00297 (Pharmacia Biotech AB) discloses arecombinant form of protein A, wherein a cysteine residue has been addedto the protein A molecule to improve its coupling to a separation matrixfor subsequent use as an affinity ligand. Further, U.S. Pat. No.6,197,927 (assigned to Genentech Inc.) discloses Z domain variants ofStaphylococcal protein A exhibiting an IgG-binding capacity equivalentto the wild type Z domain, but a significantly reduced size. However,the binding properties of protein A are not ideal. As is well known,protein A binds to IgG molecules from various mammals, with the highestaffinity to the human subclasses of IgG1, IgG2 and IgG4. It bindsprimarily to a surface formed at the juncture of both the second and thethird constant domains, known as CH2 and CH3, of IgG located on the Fcfragment. Consequently, protein A cannot be used in affinitypurification of any other fragments of IgG than Fc-containing fragments.In addition, even though protein A binds to some Fab fragments, thisbinding is not generic, since it targets the variable region. However,the interest in Fab and F(ab′)₂ fragments has increased lately, sincethey are smaller than intact IgG molecules but still contain thefunctional antigen-binding region. Accordingly, the above-mentioned lackof generality becomes another drawback with protein A ligands. Moreover,in attempts to purify IgGs of subclass 3 with protein A-ligands,problems have been reported due to a precipitation of the IgG3 whichprecipitation is irreversible, thereby causing a loss of purifiedantibody. Furthermore, protein A exhibits some further drawbacks relatedto its being a protein. Like most proteins, it is amenable toproteolytic degradation, which may pose serious problems e.g. if a celllysate is directly applied to a column comprising protein A-basedligand, since most cell lysates will also comprise various proteases.Further, protein A-based ligands are usually labile to theconventionally used cleaning in place (cip) procedures at high pHconditions, which renders reuse of the column more difficult. Inaddition, protein A-based affinity ligands have also been known to beunstable under acidic conditions, which may result in an undesiredleakage of the ligand during the purification process which will bothcontaminate the product and impair the quality of the purificationsystem.

Another ligand suggested for use in affinity chromatography has beendisclosed in U.S. Pat. No. 4,977,247, namely the cell wall protein knownas protein G. More specifically, protein G exhibits a different affinityto IgGs as compared to protein A. Protein G binds to a highly conservedregion of the constant part of the Fab fragment, primarily to residuesfrom the heavy chain, and consequently it has potential to be used as ageneric Fab binder. However, it has been reported that protein G has areduced binding to Fab fragments of type IgG2. In addition, protein Gshares most of the disadvantages of protein-based affinity ligandsdiscussed above in relation to protein A. Furthermore, many of the knownprotein-based affinity ligands have proven to be relatively expensive toproduce.

Consequently, there is a need of novel IgG-binding ligands of a moreadvantageous nature, which are also more cost-effective to produce. Suchnew ligands should avoid the above-discussed drawbacks, and preferablyalso involve more preferable binding properties than the hithertosuggested ligands.

In a recent work by the present inventors, which at the time of filingof the present patent application was still not published, a novelbinding site that exhibits the spatial conformation of a pocket wasidentified. The binding pocket was shown to be specific for human kappaIgGs of all subtypes.

The recently identified binding pocket directed the present inventors toa new target on the human IgG molecule in their efforts to find a newaffinity ligand with improved properties as compared to the prior art.

SUMMARY OF THE PRESENT INVENTION

One object of the present invention is to provide a novel ligand tohuman IgG-molecules of κ-type, which avoids one or more of theabove-discussed disadvantages.

A specific object of the present invention is to provide a novel ligandto human IgG-molecules of κ-type, which is general for all subclasses ofsaid IgGs.

Another object of the invention is to provide a novel ligand to humanIgG-molecules of κ-type, which is capable of specific binding to saidIgGs.

Yet another object of the present invention is to provide a novel ligandto human IgG-molecules of κ-type, which conforms spatially with abinding pocket defined by the amino acids of the interacting surfacesdefined in FIG. 2, or with essential parts thereof.

An additional object of the present invention is to provide a novelligand to human IgG-molecules of κ-type, which exhibits moreadvantageous chemical properties than protein-based affinity ligandse.g. at extreme pH values and which is more cost-effective to produce.

Further objects and advantages of the present invention will appear fromthe detailed description that follow below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the executed synthetic route to variations of thesubstitution pattern of a compound according to the invention and alsooutlines how in the experimental part below, the compounds in thedirected library were provided with a handle for immobilisation.

FIG. 2 shows orthographic views of the herein-discussed binding pocketin chicken net model.

FIG. 3 shows a selection of compounds according to the invention,wherein the substitution pattern has been varied.

FIG. 4 shows orthographic views of some of the compounds derived fromAB_(—)0001250.

FIG. 5 A-E show orthographic views of the docked compoundsAB_(—)000125[1-5].

FIGS. 6A and B show the structure coordinates of the amino acids thatform the interacting surfaces of a binding pocket, which is specific forhuman IgGs of κ-type. Said binding pocket, and compounds comprising saidinteracting surfaces, were identified by the present inventors andclaimed in a separate patent application, which was still pending, butnot public at the time of the present filing.

FIG. 7 shows the results of affinity chromatography on a separationmatrix according to the invention, wherein a Fab-fragment of κ-type issuccessfully isolated.

FIG. 8 shows the results of affinity chromatography on a separationmatrix according to the invention, wherein another Fab-fragment ofκ-type is successfully isolated.

FIG. 9 shows as a comparative test an attempt to isolate a Fab-fragmentof lambda-type by affinity chromatography on a separation matrixaccording to the invention.

DEFINITIONS

The terms “antibody of κ type”, “Fab fragment of κ type” and “F(ab′)₂fragment of κ type” mean herein an antibody, a Fab fragment and anF(ab′)₂ fragment respectively, wherein the light chain is of κ type.

The term “ligand” means herein a chemical entity capable of specificbinding to a target.

The term “associating with” refers to a condition of proximity between achemical entity, or portions thereof, and a target i.e. a binding pocketor binding site on a protein. The association may be non-covalent,wherein the juxta-position is energetically favoured by hydrogen bondingor van der Waals or electrostatic interactions, or alternatively it maybe covalent.

The term “functional derivative” is used to mean a chemical substancethat is related structurally and functionally to another substance.Thus, a functional derivative comprises a modified structure from theother substance, and maintains the function of the other substance,which in this instance means that it maintains the ability to interactwith the same ligands. Thus, a “functional derivative” can be either anatural variation or fragment thereof, or a recombinantly producedentity. In addition, a “functional derivative” can also comprise addedmolecules or parts, as long as the described function is essentiallyretained.

The term “binding pocket”, as used herein, refers to a region of amolecule or molecular complex, that, as a result of its hollow shape,favourably contributes to the molecule's association with anotherchemical entity. The term “interacting surface” means herein a surfacecomprised of residues capable of interacting with a binding molecule orother entity, e.g. by ionic attraction, hydrogen bonds, Van der Waalsinteraction etc.

The term “strictly conserved” is used herein to mean that after asequence alignment of all sequences available from an internationallyrecognised sequence database, the residue type is exactly the same at aspecific position for all aligned sequences. An example of such adatabase is the non-redundant database provided by the National Centrefor Biotechnology Information.

The term “structure coordinates” refers to Cartesian coordinates derivedfrom for example mathematical equations related to the patterns obtainedon diffraction of a monochromatic beam of X-rays by the atoms(scattering centres) of a protein or protein-ligand complex in crystalform. The diffraction data are used to calculate an electron density mapof the repeating unit of the crystal. The electron density maps are thenused to establish the positions of the individual atoms of the proteinor protein complex.

A “pharmacophore” is defined herein as the assembled atoms or centres ina target molecule, which have critical interactions with a receptor.Some types commonly used include hydrogen bond donors; hydrogen bondacceptors; positively or negatively charged centres; aromatic ringcentres; and hydrophobic centres.

The term “docking” means herein a fitting operation, wherein the abilityof a chemical entity to bind or “dock” to a binding site is evaluated.

The term “library” means a collection of molecules or other chemicalentities with different chemical structures and/or properties.

The term a “Conolly surface” defines the surface of the volumeaccessible to a hard spherical probe of a given radius, usually taken as1.4 Å, which is the radius of water in ice form. This surface can beobtained by “rolling the probe” over the atoms of the protein.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is a compound capable ofassociating with human IgGs of κ-type and functional derivativesthereof. More specifically, the present compound is capable of specificand reversible binding to a binding pocket of a human IgG of κ-type,which binding pocket is defined by the structure coordinates for theamino acids that constitute the interacting surfaces as shown in FIG. 6.Said binding pocket was recently identified by the present inventors,and is located between the two domains (CH1 and CL) of the constant partof κ-Fab. Thus, the herein discussed binding pocket provides a novelbinding site for human IgGs of 1-type, which binding site is a generalbinding site for all such IgGs as well as fragments or functionalderivatives thereof.

The present invention is based on an evaluation of a large number ofpotential binders to κ-Fab of human IgGs, wherein virtual screening hitswere tested with NMR. The results from the NMR was subsequently utilisedto derive structure-activity relationships that led to the constructionof a pharmacophore, and a library of affinity ligands was then designedto optimise binding and include a handle for immobilisation to achromatographic support. As will be disclosed in detail in theExperimental part below, the present inventors have studied differentsubstitution patterns and evaluated a wide range of structures in orderto identify the features required for a compound to exhibit asatisfactory binding to human IgGs of κ-type via the above discussedbinding pocket.

More specifically, the compound according to the invention is based onan N,N-alkylated urea moiety located between an aromatic part and analiphatic part. In the most preferred embodiment, the present inventionis an IgG-binding compound represented by formula (I) below

wherein

-   R₁ is CH₃ or CH₂CH₃;-   R₂ is a para and/or meta substituted phenyl group;-   R₃ is H, CH₃ or CH₂CH₃; and-   R₄ is a linear or cyclic aliphatic group, which is optionally    substituted, or, wherein-   R₁ and R₂ are as stated above while R₃ and R₄ are both parts the    same 4- to 6-membered cyclic entity, which is optionally    substituted,    and which compound has affinity for human IgG of κ-type.

Thus, in one embodiment, the compound is an affinity ligand withaffinity for a Fab fragment of human IgG of κ-type. In some contexts,such an affinity ligand is denoted an affinity adsorbent or an antibodyadsorbent.

As the skilled person in this field will easily appreciate, in formula(I), the bonds between the carbonyl carbon and each one of the nitrogenatoms are rotatable. Consequently, position R₁ is equivalent to positionR₂ and position R₃ is equivalent to position R₄, and the definitionsherein will encompass any definition of a compound, wherein R₁ has beeninterchanged with R₂ as well as when R₃ has been interchanged with R₄.Likewise, because of the inherent symmetry around the keto group, thepair R₁/R₂ is interchangeable with the pair R₃/R₄ so all thesedefinitions are also included.

In an advantageous embodiment of the compound, in formula (I), R₁ isCH₃.

As mentioned above, in formula (I), R₂ is a phenyl group, which may besubstituted with one or two halogens, such as F, Cl, Br, or I. Sincesubstituents in ortho position have been observed to have a negativeimpact on binding, any substituents are present in meta and/or paraposition. Thus, in a specific embodiment, R₂ is substituted with Cl or Fin the meta position. In another embodiment, R₂ is substituted with Clin the meta position and F in the para position. In another embodiment,R₂ is substituted with F in the meta position and Cl in the paraposition. In yet another embodiment, R₂ is substituted with Cl in metaand para position.

Alternatively, or additionally, the R₂ phenyl group is substituted withone or more oxygen-comprising groups. Thus, in one embodiment, R₂ is asubstituted phenyl group and the substituents are selected from thegroup that consists of F, Cl, Br, I and OH, preferably F and Cl.

In a specific embodiment, R₂ is substituted in the para and/or metaposition with a group defined as —O—R₅, wherein R₅ is CH₃ or CH₂CH₃, andpreferably CH₃.

As appears from the modelling described in the experimental part below,when the present compound binds to an IgG molecule, R₂ will be locatedin the inner part of the pocket and hence interact with the inner aminoacids of the interacting surfaces of the binding pocket. Largerring-systems than six-membered rings were according to NMR screeningdescribed in the experimental part below found to have a negativeinfluence on binding, and are hence avoided. Also, as mentioned above,in the most preferred embodiment, the aromatic group does not compriseany heteroatoms, since especially the presence of nitrogen atom(s) inthe ring has been observed to have a negative impact on binding.However, in an alternative embodiment, the invention is a compoundrepresented by the chemical formula (I) as defined above, wherein R₂ isanother aromatic group than phenyl. In the most preferred embodiment ofthis alternative, R₂ comprises thiophene.

As mentioned above, R₃ can be H or CH₃ or CH₂CH₃

As mentioned above, in formula (I), R₄ can be a linear or cyclicaliphatic group, which is substituted or unsubstituted. In this context,an aliphatic group can be any linear or branched carbon chaininterrupted by any heteroatom, as long as the compound fits sufficientlywell in the herein-defined binding pocket to provide binding thereof. Inone embodiment, the aliphatic chain comprises one or more carbonylgroup(s).

In one alternative embodiment, R₄ is an aromatic group that comprises aphenyl group. In one embodiment, said phenyl group is substituted in theortho and/or meta and/or para position. In a specific embodiment, saidphenyl group comprises one or more heteroatoms, such as N, S etc.

In a specific embodiment, R₄ can be a methyl-substituted amino acidresidue, or a derivative thereof. Thus, in a specific embodiment, R₄ isselected from the group that consists of aliphatic amino acid residues,hydroxyl-containing amino acid residues, sulphur-containing amino acidresidues, aromatic amino acid residues, acidic amino acid residues,basic amino acid residues or imino-containing amino acid residues, orany derivative thereof.

In a specific embodiment, which is especially advantageous if thecompound is to be used in a form immobilised to a solid support, e.g. asa ligand in affinity chromatography, the aliphatic group R₄ alsocomprises terminating functionalities useful for such immobilisation.Thus, in one embodiment, an aliphatic group is a linear or branchedcarbon chain as discussed above, which is terminated with a carboxylicacid i.e. —COOH. In an alternative embodiment, the aliphatic group isterminated with a carboxylic acid derivative, such as an ester, ahalide, an amide, a nitrile or the like. In an alternative embodiment,an aliphatic group is a linear or branched carbon chain as discussedabove, which is terminated with nitrogen, oxygen, sulphur or anyderivative thereof. Such derivatives are well-known to the skilledperson in this field, and are also useful for immobilisation. Asmentioned above, the only limitation in this context is that thealiphatic group does not impair the binding of the compound to theherein defined binding pocket.

In another embodiment, in formula (I), R₄ is CH₃. In a specificembodiment, both R₃ and R₄ are CH₃. In a specific embodiment, in formula(I), R₁ is CH₃; R₂ is a phenyl group that has been substituted with Clin meta and para position; R₃ is CH₃; and R₄ is CH₃. In one embodiment,the present compound is selected from the compounds shown in FIG. 3.

As also appears from the above, in an alternative embodiment, R₃ and R₄are parts of a 4- to 6-membered cyclic entity. In an advantageousembodiment, the cyclic entity is 3- to 5-membered. Consequently, saidcyclic entity comprises the N of Formula (I), R₃ and R₄ and optionally 1or 2 other atoms, which may be carbon atoms or heteroatoms. In the mostpreferred embodiment, the R₃ and R₄ substituents constitute an aminoacid derivative. In one embodiment, R₃ and R₄ are part of a 5-memberedcyclic entity, which in turn is substituted, preferably with a groupuseful for immobilisation as discussed above. In a specific embodiment,a 5-membered cyclic entity is substituted in the position adjacent tothe N with a C(O)—O—CH3 group, and consequently the R₃ and R₄substituents of this embodiment are parts of a D-proline derivative.This specific embodiment is denoted AB_(—)0003290 in FIG. 3.

Furthermore, the present invention also encompasses a compound, which isbasically represented by formula (I) above, but wherein R₁ and R₃ arecarbon atoms connected to each other to form a cyclic structure. In thisembodiment, R₃ is a carbonyl group. In this embodiment, R₄ is preferablya phenyl group. Thus, this embodiment of the compound is known as1,3-diphenylimidazolidine-2,4-dione.

In order to provide the best binding to the herein-discussed bindingpocket of a human IgG of κ-type, or to a functional derivative thereof,it is preferable that the compound has a non-planar geometry. In thecontext of the binding pocket, it is noted that the present compound iscapable of binding to binding pockets not only of the exact definedstructure coordinates as defined herein, but also to pockets defined byinteracting surfaces having a mean square deviation from the backboneatoms of the disclosed binding pocket amino acids of not more than 2.0Å. In a preferred embodiment, said deviation is not more than about 1.5Å and in the most preferred embodiment, said deviation is not more than1.0 Å. In one embodiment, the present compound is capable of binding toa human IgG or a functional derivative thereof with a binding constantof at least 10⁻³ M, preferably at least 10⁻⁶ M and most preferably atleast 10⁻⁸ M. Thus, illustrative intervals of such binding are e.g. 10⁻³M 1 to 10⁻⁸ M, such as 10⁻³ M⁻⁴ to 10⁻⁶M or 10⁻⁶ to 10⁻⁸M.

In a specific embodiment, the present compound is capable of binding toa human IgG of κ-type, or a functional derivative thereof, via a bindingpocket formed between two polypeptides, wherein the first polypeptide isthe portion of a human IgG κ light chain that starts at one of aminoacids 93 to 110 and ends at one of amino acids 187 to 214 of human IgG κlight chain and the second polypeptide is the portion of a human IgGheavy chain that starts at one of amino acids 106 to 128 and ends at oneof amino acids 215 to 225 of human IgG heavy chain. In the herein usedenumeration of amino acids refers to a human sequence wherein no. 93 isthe first amino acid of the constant domain, as also used in FIG. 6.

Thus, the IgG-binding compounds according to the present invention arein general smaller than the prior art affinity ligands used for antibodyisolation. In addition, the compounds according to the invention areorganic molecules that lack the peptide structure of e.g. protein A- andprotein G-based ligands, which in general renders them less susceptibleto extreme pH values. Naturally, they are not as susceptible toproteolytic degradation, or any other kind of degradation, as theprotein-based prior art ligands either. In addition, the presentcompounds are more cost-effective to produce.

The compound according to the invention can be prepared by the skilledperson in this field using well-known methods, as illustrated e.g. inFIG. 1 below and as explained in the experimental part below under“Synthesis”.

A second aspect of the invention is the use of a compound as definedabove for selective binding of human IgG of κ-type, or a functionalderivative thereof. In the present context, it is understood that theencompassed derivatives can be any human κ-Fab constant part-comprisingcompounds, i.e. any composition comprising the globular region of an IgGmolecule formed by the first constant domain of the heavy chain (CH1)and the constant domain of the light chain (CL). Thus the term includesany of the following terms which are well known from standard IgGterminology: Intact IgG molecules, F(ab′)₂ fragments, Fab′ fragments,Fab fragments and by definition the globular region named itself, all ofwhich have human sequences and light chains of κ-type. This definitionincludes also any modifications of named IgG or named antibody fragmentsincluding even chimeric molecules formed in one part of one of saidcompositions and in another part of any of the following proteins,peptides, carbohydrates, lipids or any other organic or inorganic entityand chimeric combinations thereof and also any of the above-mentionedcovalently attached to solid phase.

The present invention also encompasses a sorption complex comprised of acompound as defined above directly linked to the Fab fragment of a humanIgG of κ-type, or a functional derivative thereof. More specifically,the compound is linked to the Fab fragment of said antibody, and morespecifically to the herein described binding pocket. Such a sorptioncomplex will form as the compound according to the invention iscontacted with a solution comprising human IgG's of κ-type, or afunctional derivative thereof, under suitable conditions. The skilledperson in this field can easily select such conditions and adjust pH,ionic strength etc to provide or to break up the complex.

Another aspect of the invention is a separation matrix for use inaffinity chromatography, wherein the ligands comprises at least onecompound as defined above. In a specific embodiment, the ligands havebeen coupled to a support via linkers. The present matrix can e.g. be inthe form of separate particles, preferably porous and essentiallyspherical particles; a monolith; or a membrane.

The present invention also encompasses a system suitable for affinitychromatography, which is comprised of a separation matrix as definedabove packed in a column. The column may be of a size suitable foranalytical scale or for large scale chromatography.

Suitable support materials are well known. In one embodiment, thesupport is a natural polymer, such as agarose, alginate, carrageenan,gelatine etc. Such natural polymers are known to form physicallycross-linked networks spontaneously on cooling or on addition ofdivalent metal ions, and chemical cross-linkers can be added if desired.This kind of supports is easily prepared according to standard methods,such as inverse suspension gelation (S Hjertén: Biochim Biophys Acta79(2), 393-398 (1964). In another embodiment, the support is comprisedof cross-linked synthetic polymers, such as styrene or styrenederivatives, divinylbenzene, acrylamides, acrylate esters, methacrylateesters, vinyl esters, vinyl amides etc. Such polymers are also easilyproduced according to standard methods, see e.g. “Styrene based polymersupports developed by suspension polymerization” (R Arshady: Chimica eL'Industria 70(9), 70-75 (1988)). Thus, in summary, the support materialcan in principle be any material that allows the covalent coupling ofthe IgG-binding compound discussed above, such as the above-discussedpolymers, inorganic materials, such as silica, ceramics etc.

Many well-known methods are available for immobilising ligands to asupport through suitable functional groups. As the skilled person inthis field will realise, the exact choice of coupling method will dependon the structure of the ligand to be immobilised. In one embodiment, thesupport has hydrophilic surfaces, and if porous, the surfaces of thepores are also hydrophilic. This is advantageous in order to avoid or atleast reduce any non-specific protein interactions. It is alsoadvantageous if the surfaces have a high density of groups available forcoupling of ligands. Such coupling groups are commonly hydroxyl groups,but may also be allyl groups i.e. double bonds available for grafting,amines, thioles, epoxides and the like. If the support material hasundesirable surface properties, it is possible to coat it with ahydrophilic polyhydroxy-functional material before coupling the ligand.The techniques and considerations for coupling of affinity ligands to asuitable support to prepare a separation matrix are well known in thisfield, see e.g. WO 98/33572 for a detailed review of coupling chemistryas well as suitable linking molecules, therein denoted “extenders”.

Another aspect of the invention is a generic method of isolating orseparating a target compound, i.e. a human IgG of κ-type, or afunctional derivative thereof, from other components in a liquid,wherein a compound or a separation matrix as defined above is used. Inthe context of immunology, the separation matrix is often denoted an“immunsorbent”. In the most preferred embodiment, the present method isaffinity chromatography, which is a widely used and well-knownseparation technique. In brief, in a first step, a solution comprisingthe desired antibodies is passed over a separation matrix underconditions allowing adsorption of the antibody to ligands present onsaid matrix. Such conditions are controlled e.g. by pH and/or saltconcentration i.e. ionic strength in the solution. Care should be takennot to exceed the capacity of the matrix, i.e. the flow should besufficiently slow to allow a satisfactory adsorption. In this step,other components of the solution will pass through in principleunimpeded. Optionally, the matrix is then washed, e.g. with an aqueoussolution, in order to remove retained and/or loosely bound substances.In a next step, a second solution denoted an eluent is passed over thematrix under conditions that provide desorption i.e. release of thedesired antibody. Such conditions are commonly provided by a change ofthe pH, the salt concentration i.e. ionic strength, hydrophobicity etc.Various elution schemes are known, such as gradient elution andstep-wise elution. Elution can also be provided by a second solutioncomprising a competitive substance, which will replace the desiredantibody on the matrix.

In an alternative embodiment, the compound according to the invention isused in site-specific modification of a human IgG of κ-type, or afunctional derivative thereof. More specifically, a human IgG of κ-type,or a functional derivative thereof, can be modified by binding acompound as defined above selectively to the binding pocket identifiedby the present inventors. In a specific embodiment, the modification isa stabilisation of Fab-folding.

In an alternative embodiment, the present compound is used in animmunological assay for detection of a human IgG of κ-type, or afunctional derivative thereof. In this case, the compound is preferablylabelled with a suitable detectable label as conventionally used, suchas a fluorescent label, a luminescent label, a chemiluminiscent label,an enzyme label, a radioactive label, an absorbance label etc. Suchassays may be in solution or on solid phase. In one embodiment, thehuman κ-Fab constant part-comprising composition is a human IgG or afragment thereof. In the preferred embodiment, the present assay is acompetitive assay, wherein the ability of a candidate ligand to displacea known ligand's binding to a compound or binding pocket as definedabove is evaluated.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the executed synthetic route to i) variations of thesubstitution pattern and ii) provide the compounds in the directedlibrary with a handle for immobilisation as discussed below in Example2. To the top-left, the original hit AB_(—)0001250 is shown. Thesynthesis will be described in detail below in the section Materials andmethods.

FIG. 2 shows orthographic views of the herein-discussed binding pocketin chicken net model. The amino acid residues forming the pocket areshown in stick model and the corresponding structure coordinates arepresented in FIG. 6. Docked hit AB_(—)0001250 is shown in space-fillmodel to illustrate the possibilities of the pocket to harbour asubstituted phenyl ring.

FIG. 3 shows a selection of compounds according to the invention,wherein the substitution pattern of R₁, R₂ as well as R₃ and R₄ has beenvaried. A central N,N-alkylated urea moiety as well as a para and/ormeta substituted phenyl groups are present in all the compounds.

FIG. 4 shows orthographic views of compounds derived from AB_(—)0001250.Five docked hits superimpose very well onto the original hitAB_(—)0001250.

FIG. 5 A-E show orthographic views of the docked compoundsAB_(—)000125[1-5] in the binding pocket, as discussed in more detail inthe experimental part below.

FIGS. 6 A and B show the structure coordinates of the amino acids thatform the interacting surfaces of the binding pocket shown in FIG. 2,which is specific for human IgGs of κ-type. FIG. 6A shows thecoordinates of the light chain, while FIG. 6B shows the heavy chain.More specifically, the structure coordinates shown form a small pocketin between the two domains (CH1 and CL) of the constant part of κ-Faband constitutes a novel target binding site. The residues forming thepocket together with some residues located at the entrance andcontributing significantly to the topology of the putative binding sitehave been identified as follows. From the light chain, there are Q124,S127, G128, T129, S131, V133, G157, N158, S159, Q160, E161, S162, S176,S177, T178, T180, L181, and they are all strictly conserved for allsequences of κ-type identified in a sequence homology search. Theresidues from the heavy chain are K126, P128, S129, F131, L133, L150,K152, D153, F175, P176, V178, L179, Q180, S181, S182, L184, S186, L187and S188, bold being strictly conserved and remaining highly conserved.The structure coordinates of the full amino acid sequence of a human IgGof κ-type can be obtained from the Protein Data Bank, accession code1vge, e.g. at—http://www.rcsb.org/pdb/.

FIG. 7 illustrates how a Fab-fragment of a monoclonal antibody ofkappa-type can be isolated by affinity chromatography using a separationmatrix according to the invention, as described in Example 6 below.Injection of the monoclonal ABFab-K1 on the AB_(—)0003291-containingmedium according to the invention in PBS, 1 M (NH₄)₂SO₄, pH 7 at 0 ml.Cleaning in place (CIP) starts at 10.0 ml. The small peak at 1.07 ml isdue to injection effect. The protein is not totally removed from thecolumn during the CIP. Evidently, the affinity column is able to bindthe monoclonal ABFab-K1.

FIG. 8 illustrates how another Fab-fragment of a monoclonal antibody ofkappa-type can be isolated by affinity chromatography using a separationmatrix according to the invention, as described in Example 6 below.Injection of the monoclonal ABFab-K2 on the AB_(—)0003291-containingmedia in PBS, 1 M (NH₄)₂SO₄, pH 7 at 0 ml. CIP starts at 10.0 ml. Thesmall peak at 1.09 ml is due to injection effect. The protein is nottotally removed from the column during the CIP. Evidently, this affinitycolumn is also able to bind the monoclonal ABFab-K2, and the compoundaccording to the invention can consequently be described as a liganduseful as a general binder of human IgG Fab fragments of κ-type.

FIG. 9 illustrates as a comparative test how a Fab-fragment of amonoclonal antibody of lambda-type is tested in affinity chromatographyon a separation matrix according to the invention, as described inExample 6 below. Injection of the monoclonal ABFab-L2 on theAB_(—)0003291-containing media in PBS, 1 M (NH₄)₂SO₄, pH 7 at 0 ml. CIPstarts at 10.0 ml. From this figure, it clearly appears that theABFab-L2 directly comes off the affinity column and is found in theflow-through. Accordingly, the separation matrix according to theinvention is not suitable for isolation of Fab-fragments of lambda-type,and confirms the statement above that the compound according to theinvention is a binder of human IgG Fab fragments of κ-type, but not oflambda-type.

Experimental Part

Below, the present invention will be explained in more detail by way ofexamples, which however are not to be construed as limiting the presentinvention as defined by the appended claims. All references given belowand elsewhere in the present specification are hereby included herein byreference.

Materials and Methods

Molecular Modelling

Compounds of the directed library were sketched with MDL ISIS/draw andtransferred to an OCTANE™ (Silicon Graphics Inc.®) workstation providedwith two 195 MHz R10000 processors. The program package SYBYL® (TriposInc., 2000) was used for all remaining modelling.

Preparation of Compounds for Docking

The structures of the compounds were transformed into 3D using theprogram CONCORD and ionised to reflect their most probable protonationstate at pH 7. The coordinates were then subject to 500 cycles ofminimisation using the MMFF94 force field (Halgren 1996—Halgren, T.1996. Merck molecular force field. I. Basis, form, scope,parameterisation, and performance of MMFF94. J. Comp. Chem. 17:490-519.).

Docking of Prepared Molecules

Docking simulations have been performed with the program FlexX™ (Rareyet al. 1996 Rarey, M., Kramer, B., Lengauer, T., and Klebe G. 1996. Afast flexible docking method using an incremental constructionalgorithm. J. Mol. Biol. 261: 470-489.) which is part of the SYBYLpackage. FlexX allows flexibility in the ligands, keeping the receptorfixed. All the relevant receptor information necessary for the dockingsimulations is stored in the receptor definition file (rd file). FlexXuses formal charges, which were turned on during the dockingsimulations. The protein structure used was the highest-resolution (2.0Å) crystal structure of κ-Fab (accession code to the Protein Data Bank1vge, Chacko et al., 1996 Chacko, S., Padlan, E. A., Portolano, S.,McLachlan, S. M., Rapoport, B.: Structural studies of humanautoantibodies. Crystal structure of a thyroid peroxidase autoantibodyFab. J Biol Chem 271 pp. 12191 (1996)). The following residues wereincluded in the definition of the binding site: from the light chain:Ser-131, Val-133, Ser-159, Gln-160, Glu-161, Ser-162, Ser-176, Thr-178,and Thr-180. From the heavy chain: Leu-150, Lys-152, Phe-175, Pro-176,Val-178, Gln-180, Ser-186, Leu-187, Ser-188. All of these residues havepreviously been shown by the present inventors to be strictly conservedas observed from a sequence alignment and are a subset of the identifiedpocket. The subset was created by taking all residues with at least oneatom at a distance of at least 4 Å from the docked hit AB_(—)0001250 andsubsequently by including some additional residues to complete a Conollysurface of the pocket surrounding the docked hit. In the proteinstructure, the ε carbonyl oxygen of H:Gln-180 is located 2.5 Å away fromone of the δ carboxyl oxygens of H:Asp153. This was assumed to be anerror due to misinterpretation of the electron density of thecarboxyamide terminal group of H:Gln-180, and the group was consequentlyflipped around 180°. In this corrected structure, the ε nitrogen ofGln-180 from the heavy chain is at favourable hydrogen bonding distanceto the carboxyl oxygen of H:Asp153. Otherwise, defaults have been usedwhen creating the rd file and no special customisations have been done.When necessary the SYBYL LINE NOTATION (sin) core option of FlexX inSYBYL was applied to bias the docking towards conformations that werecompatible with the expected binding mode with the phenyl ring insidethe pocket. The sin core option was applied with inputN(C(NCH3)=O)(C[9]:CH:CH:C:C:CH:@9)CH3 to indicate to the program tostart fragment build-up using a common substructure of the six compoundsin the directed library. Prior to docking, all water molecules wereremoved. The 30 best ranked conformations and their FlexX score weresaved for each molecule.

Synthesis of Library Based on AB_(—)0001250 Synthesis of 4-(methylamino)butyric acid methyl ester

4-(methylamino) butyric acid HCl was dissolved in methanol and thionylchloride in catalytic amount was added drop by drop. The reactionmixture was stirred at 0° C. for 30 min. Thereafter, the solvent wasreduced in vacu, yielding a white solid.

Synthesis of 3,4-dichloro-/(N-methyl)-aniline

3,4-dichloro aniline (40 mmol, 5 g) was dissolved in 400 mL of DCM. Tothis solution was added iodo methane (40 mL), triethyl amine (5 mL), andNaH (40 mmol, 3.8 g). The resulting mixture was stirred at ambienttemperature over night, where after small aliquots of water summing upto a total of 50 mL of water was added, followed of an additional hourof stirring. The reaction mixture was transferred to a separation funneland extracted with 5% sodium thio-sulphate, dried over magnesiumsulphate and concentrated in vacu to almost complete dryness. Thematerial was separated by silica chromatography (pentane:ether—8:2), theappropriate fractions were collected and concentrated in vacu to almostcomplete dryness, yielding 3 g of material including some solvent. Thecorrect material was indicated by LC-MS analysis. This material wasdirectly used in the subsequent step.

General Method for Synthesis of N-methylated Aniline Derivatives

The aniline derivative was dissolved in DCM and sodium hydride (in thecase of AB_(—)0001253 sodium bis(trimethylsilyl) amide) (1.5 eq) anddi-tertbutyl-di-carbonate (1.3 eq) was added followed by stirring atroom temperature over night. The reaction mixture was transferred to aseparatory funnel and extracted with water, dried over magnesiumsulphate, and concentrated in vacu. The crude product was dissolved inTHF and lithium alumina hydride (1.2-2 eq) was added and the reactionmixture was refluxed until completion as indicated by LC-MS. Thereafterthe mixture was filtered. This filtrate was used directly in thesubsequent step.

General Method for Synthesis of Urea Derivatives

To a THF solution of the N-methylated aniline (or the non-N-methylatedaniline derivative) was added phosgene (20% in toluene) in large excessand the reaction mixture was stirred at room temperature for 30 min,concentrated in vacu, and re-dissolved in DCM. To this solution wasadded an excess of triethyl amine and 4-(methylamino) butyric acidmethyl ester (or 4-amino butyric acid methyl ester) (approx. 1 eq). Thereaction mixture was stirred at room temperature for 3 hours,concentrated in vacu, and purified by RP-HPLC.

General Method for Hydrolysis of Methylesters

The methyl ester of the urea derivative (0.5 g) was dissolved inmethanol (10 mL) and lithium hydroxide (0.25 g) was added. The resultingmixture was stirred at ambient temperature for 5 hours, neutralised with1 M HCl, and concentrated in vacu. The resulting material was purifiedby RP-HPLC.

Synthesis of 1-(3,4-dichlorophenyl)-1,3-dimethyl-3-butyric acid urea

3,4-dichloro-N-methyl-aniline (all material from previous description)was dissolved in 200 mL of DCM. To the solution was added phosgene (20mL, 20% sol. in toluene) and the mixture was stirred for 30 minutes atambient temperature. The solvent was removed in vacu and an additional100 mL of DCM was added, followed by removal of the added solvent invacu.

The remaining solid was dissolved in 200 mL of DCM and 4-methyl-4-aminobutyric acid (2 g) was added followed by the addition of triethyl amine(5 mL). The resulting mixture was stirred at ambient temperature during2 hours. Thereafter, the reaction mixture was transferred to aseparation funnel and partitioned between DCM and water. The organicphase was isolated, dried over magnesium sulphate, and concentrated invacu. The remaining material was purified by silica columnchromatography (DCM:Et-OH—9:1), the appropriate fractions were collectedand concentrated in vacu to yield 1.4 g of the desired material as aclear oil.

EXAMPLE 1 Binding Test Using NMR

All NMR experiments were performed at 298 K on a Bruker Avance 500 MHzspectrometer. The 1D saturation transfer difference method (STD-NMR) wasused as screening assay (Mayer M. and Meyer B. 1999. Characterisation ofLigand Binding by Saturation Transfer Difference NMR Spectroscopy.Angew. Chem., Int. Ed. 38: 1784-1788). The resulting STD-NMR spectrumshows the difference between spectra recorded with on- and off-resonanceirradiation of the protein, respectively. The two spectra are recordedin the same experiment in an interleaved fashion. If the resultingSTD-NMR spectrum shows the same signals as the reference ¹H-NMR spectrumof the ligand the result is regarded as positive i.e. the ligand musthave contacted the protein. Ligands that do not have any contact withthe protein or are very tightly bound to the protein will not give anysignal in the resulting STD-NMR spectrum. It has been shown that themethod is capable of detecting ligands with dissociation constantsbetween 10⁻³ and 10⁻⁸ M (Mayer M. and Meyer B. 1999. Characterisation ofLigand Binding by Saturation Transfer Difference NMR Spectroscopy.Angew. Chem, Int. Ed. 38: 1784-1788). The strength of the STD-NMR signaldepends upon several factors including protein size, offset and durationof the on-resonance irradiation, the dissociation rate constant and theexcess of ligand. The STD-NMR method is advantageous in that thedetection limits can be tuned for binding by varying the proteinconcentration while keeping the ligand concentration constant. Undersuch conditions, at higher protein concentrations the weak to mediumbinders are detected, whereas at lower protein concentrations onlymedium binders are detected. For instance, it has been shown before foranother enzyme system that both μM and mM binders were detected at aprotein concentration of 35 μM whereas only μM binders were detected atprotein concentrations of 1 μM and 100 nM (Peng J. W., Lepre C. A.,Fejzo J., Abdul-Manan N. and Moore J. M. 2001, Nuclear MagneticResonance-Based Approaches for Lead Generation in Drug Discovery,Methods in Enzymology. 338: 202-230). It should be noted that the signalintensity at one specific protein concentration should not be taken as adirect measure of the binding strength. For instance, in the same study,a mM binder showed a stronger signal as compared to a μM binder at 35 μMprotein concentration whereas when the protein concentration was reducedto 1 μM the signal from the weaker binder vanished. On the other handthe signal of the μM binder became even stronger than before (Peng J.W., Lepre C. A., Fejzo J., Abdul-Manan N. and Moore J. M. 2001, NuclearMagnetic Resonance-Based Approaches for Lead Generation in DrugDiscovery, Methods in Enzymology. 338: 202-230).

Here, three different antibody concentrations were used, namely, 0.5 μM,100 nM and 20 nM. The antibody used was a human Fab of κ-type. In allcases ligands were tested one-by-one. On-resonance irradiation was setat 0 ppm and off-resonance irradiation was set at 40 ppm. Irradiationtime in each scan was 2 s and 16K data points were collected with 1024scans in total. Compounds for testing were dissolved in DMSO_(d6) to aconcentration of 50 mM and 5 μL of the concentrated ligand solution wasadded to 495 μL buffer solution. The samples thus consisted of 0.5 mMligand, 20 mM phosphate buffer, 100 mM NaCl and 5% DMSO_(d6) in D₂O atpD 7.5, uncorrected reading on pH-meter. Compounds were initially testedfor binding with 0.5 μM antibody. Interesting ligands were furthertested with protein concentrations of 100 or 20 nM. A one-dimensional¹H-spectrum was acquired first as reference spectrum and subsequently asaturation transfer difference (STD) spectrum was acquired. Eachanalysis took 60 minutes on the spectrometer.

The results are shown in Table 1 below, wherein the results from NMRscreening are compiled. TABLE 1 Results from the NMR screening IDChemical name conc 1 conc 2 conc 3 ctrl AB_0000510 alpha-pyridoin 0AB_0000530 1-(3-chlorophenyl)-3-methyl-2- 0 pyrazolin-5-one AB_00005401,3-diphenylparabanic acid 0 AB_0000580 n1-(3-chloro-4-fluorophenyl)-2-1 0 [(4,6-dimethylpyrimidin-2- yl)thio]acetamide AB_00006003-phenyl-1,2,4-benzotriazine 1 1 0 AB_00006102-(4-chlorophenyl)-2,3-dihydro- 0 1h-pyrrolo[3,4-c]pyridine-1,3- dioneAB_0000630 n1-(2,3,4-trifluorophenyl)-2- 0(1,2,4-oxadiazol-3-yl)acetamide AB_0000670 methyl n-[(5-methyl-4-phenyl-2 0 1,3-oxazol-2- yl)carbonyl]carbamate AB_0000690n1-(2,4-difluorophenyl)-2-(1,2,4- 0 oxadiazol-3-yl)acetamide AB_00007003,6-di-2-pyridyl-1,2,4,5-tetrazine 0 AB_00007302-benzylidene-1,3-indandione 0 AB_0000740 5-phenyl-1,2,4-oxadiazol-3-yln- 0 (4-fluorophenyl)carbamate AB_0000750 n-(5,5-dimethyl-7-oxo-4,5,6,7-0 tetrahydro-benzothiazol-2-yl)- nicotinamide AB_00007605-(2-phenyl-1,3-thiazol-4-yl)- 1 0 1,3,4-oxadiazol-2-ylhydrosulfideAB_0000790 3-[2-oxo-2-(2-pyridyl)ethyl]-1,3- 0dihydroisobenzofuran-1-one AB_0000810 2-phenoxy-2-phenyl-1-ethanol 2 1 0AB_0000860 1,3-diphenylimidazolidine-2,4- 2 2 1 0 dione AB_00008805-bromo-3-phenyl-thiazolidine- 0 2,4-dione AB_00009001-(2-naphthoyl)imidazole 0 AB_0000910 3-bromo-4-methoxyphenylacetone 2 20 0 AB_0000930 3-chloro-1-phenyl-pyrrole-2,5- 0 dione AB_00009903-butyl-2-hydroxy-4h-pyrido[1,2- 0 a]pyrimidin-4-one AB_00010003-(benzylamino)-1,1,1-trifluoro-2- 1 0 propanol AB_00010102-[5-(2-fluorobenzoyl)-2-thi- 2 2 2 1 enyl]acetonitrile AB_00010205-(3,5-difluorobenzyl)-3-(2- 1 0 thienyl)-1,2,4-oxadiazole AB_00010302-chloro-3-(trifluo- 1 0 romethyl)benzaldehyde AB_00010402-hydroxy-3-(2-methyl-1- 1 1 0 propenyl)-1,4-naphthoquinone AB_00010602-(2-imino-thiazol-3-yl)-1- 2 0 naphthalen-2-yl-ethanone AB_0001070imiloxan hydrochloride 1 1 AB_0001080 2-(benzylthio)-5-methyl-4,5- 0dihydro-1h-imidazol-3-ium chlo- ride AB_0001090n-(3-chlorophenyl)-maleimide 1 0 AB_0001100 ethyl4-oxo-1,4-dihydroquinoline- 0 3-carboxylate AB_00011303-amino-n-[2-(methylthio)ethyl]- 0 4-oxo-3,4-dihydroquinazoline-2-carboxamide AB_0001150 1-[(3,4-dichlorobenzyl)oxy]-1h- 2 0 imidazoleAB_0001170 3-[(2,4-dichlorobenzyl)amino]- 0 1,1,1-trifluoro-2-propanolAB_0001180 3-oxo-2-phenyl-2,3-dihydro-4- 0 pyridazinecarboxylic acidAB_0001190 4-[1-(2-phenylethyl)-(1h)-pyrazol- 2 0 4-yl]pyridineAB_0001200 methyl 1-hydroxy-2-naphthoate 1 0 AB_00012201-(3-trifluoromethyl- 2 0 phenyl)imidazole AB_0001230(2-naphthoxy)acetic acid, n- 0 hydroxysuccinimide ester AB_0001240methyl 3-(5-chloro-2- 1 1 0 methoxyphenyl)-2,3-epoxypropi- onateAB_0001250 1-(3,4-dichlorophenyl)-1,3,3- 2 2 1 0 trimethylureaAB_0001260 2-pyridyl 2-(2,3-dihydro-1,4- 0benzodioxin-2-yl)-1,3-thiazole-4- carbothioate AB_00012701-[(4-chlorobenzyl)amino]-3- 1 0 (phenylthio)propan-2-ol AB_00012903-[(4-chlorophenoxy)methyl]-5- 2 1 0 [(2-pyridylthio)methyl]-1,2,4-oxadiazole AB_0001300 3-(2-thienylcarbonyl)-4h- 1 0pyrido[1,2-a]pyrimidin-4-oneConcentration code as follows: conc. 1 means 500, conc 2 100 and conc 320 nM protein.NMR signal code: 0 no, 1 weak and 2 strong signal.

As regards table 1, one compound (Compound AB_(—)0001010) showed apositive NMR signal even in the absence of target antibody. Thatcompound is likely to be a false positive and was therefore excludedfrom further analysis. A total of 22 compounds did not show any bindingsignal in the NMR experiments performed at highest antibodyconcentration and were thus designated as non-binders. From 23 compoundswhich showed signal at the highest antibody concentrations a total of 14did not show any signal at the first dilution of antibody concentration.These compounds were designated as weak binders. Nine compounds showedsome kind of signal at the first dilution of antibody concentration andwere thus designated as medium to strong binders. Of these, threecompounds AB_(—)0000860, AB_(—)0000910 and AB_(—)0001250 showing a clearsignal (2 in table 1) were further analysed at a second dilution ofantibody concentration (conc 3 in table 1). Whereas compoundAB_(—)0000910 did not show any signal at this concentration, bothAB_(—)0000860 and AB_(—)0001250 did and were thus confirmed as strongbinders.

As regards the structure-activity relationships, the followingobservations arose from inspection of the structures of the compoundsbelonging to the three groups of non-binders, weak binders and medium tostrong binders. Preferable for binding seems to be the combination of anaromatic part with and aliphatic part with appropriate elements on bothparts. Positive for binding for the aromatic part is a meta- and/orpara-substituted phenyl ring without heteroatoms in the ring. Especiallythe presence of nitrogen in the ring seems to influence bindingnegatively as well as substituents in ortho position. Preferable forbinding for the aliphatic part are 1) the presence of a tertiaryanilinic nitrogen attached to position 1 and 2) (only) one β-keto groupattached to position 1, position 1 being the position where thealiphatic part of the ligands is connected to the assumed deepest layingaromatic ring. Preferably a combination of both features like in theN,N-alkylated urea moiety found in the two hits confirmed as strongbinders. AB_(—)0000860 posses two aromatic rings differing by theirrelation to the keto groups of the hydantion ring. This asymmetry givesthe molecule a direction, which from the docking analysis agrees withthe requirement of only one keto group in a β-position relative toposition 1.

The presence of two keto groups in a β-position relative to position 1disfavours binding. This is in agreement with docking results, where itcan be seen that a second keto group would probably be forced into arather unfavourable hydrophobic environment. Also, larger ring-systemsthan six member rings (for instance fused rings) seem to have a negativeinfluence for binding. Among the weak binders five compounds were foundcontaining non-substituted phenyl rings and three compounds containingtri-fluoro-methyl groups. It could be speculated that for thesecompounds the affinity detected may be related to hydrophobicinteractions of a rather non-specific type.

EXAMPLE 2 Directed Library

Actions Undertaken after the Analysis of the NMR Screening

From the analysis of the results, two directed libraries centred on thestructures of the confirmed strongest binder AB_(—)0001250 were created.

Directed Library Centred on the Structure of AB_(—)0001250

Hit AB_(—)0001250 was one of the hits designated as strong binders.Also, the structure as such offered a potentially attractive syntheticroute for varying the substitution pattern of central motif, i.e. thetetra substituted urea, including the introduction of a handle forimmobilisation, e.g. to a gel. Therefore, AB_(—)0001250 was chosen as astarting point in the continued development of improvements.

The analysis started with a search for varying the substitution patternof the aromatic ring. The di-chloro substituted aromatic ring that ispresent in AB_(—)0001250 does according to the docking fill theavailable space in an appropriate way in two dimensions but, since thatstructure is planar, a pocket above the plane of the ring was notfilled.

3-chloro-4-methoxyaniline was chosen as the starting point for furthersynthetic work, since it can be converted into the desired startingmaterial by alkylation of the anilinic nitrogen with methyl iodide.

One option is to have a fluoro-substituent in the meta position, inorder to favour hydrogen bonding of protein residues. Also for this, asuitable starting material, namely 3-chloro-4-methoxyaniline, iscommercially available. The compounds belonging to the designed directedlibrary together with the structure of the original hit AB_(—)0001250 asshown in FIG. 3 were subject to docking and NMR screening.

EXAMPLE 3 Molecular Modelling and Docking of Directed Library

The modelling and docking was performed as described above underMaterials and Methods. The results from the docking are as follows:

All docked compounds in the directed library with the exception ofAB_(—)0001256 resulted in a docked conformation inside the bindingpocket, which very closely resembles the position of the docked hitAB_(—)0001250, see FIG. 4. In four of the compounds, this is thebest-ranked solution. In one of them (AB_(—)0001252), the solutioncorresponding to the molecule inside the pocket is the second rankedsolution. AB_(—)0001256 lacks one of the methyl groups in the tetrasubstituted urea moiety. Consequently the corresponding amide bondshould be more constrained to a planar geometry as compared to theremaining compounds in the library for which such geometry is forbiddenbecause of steric effects between the methyl groups. Apparently, thenon-planar geometry is of importance for docking.

The values of the obtained expected energies of binding in kJ/mol are−10, −13, −12, −14 and −14 for AB_(—)000125 (−1) to (−5) respectively.Orthographic plots of the docked hits are shown in FIG. 5.

EXAMPLE 4 NMR Screening of Directed Library

TABLE 2 Results from NMR screening of the directed library ID Trivialname Conc 1 Conc 2 AB_0001251 4-(1,3-Dimethyl-3-phenyl-ureido)- 0 0butyric acid methyl ester AB_0001252 4-[3-(3-Fluoro-4-methoxy-phenyl)- 11 1,3-dimethyl-ureido]-butyric acid methyl ester AB_00012534-[3-(3-Chloro-4-methoxy-phenyl)- 2 1 1,3-dimethyl-ureido]-butyric acidmethyl ester AB_0001255 4-[3-(3,4-Dichloro-phenyl)-1,3- 2 2dimethyl-ureido]-butyric acid methyl ester AB_00012574-[3-(3,4-Dichloro-phenyl)-3- 2 0 methyl-ureido]-butyric acid methylester AB_0001258 4-[3-(3,4-Difluoro-phenyl)-1,3- 1 1dimethyl-ureido]-butyric acid methyl ester AB_00030904-[3-(3,4-Dichloro-phenyl)-1,3- dimethyl-ureido]-butyric acid AB_00032901-[(3,4-Dichloro-phenyl)-methyl- 2 2 carbamoyl]-pyrrolidine-2-carboxylicacid methyl ester (R-isomer)Concentration code as follows: conc. 1 means 500 and conc 2 100 nMantibody.NMR signal code: 0 no, 1 weak and 2 strong signal, nd means notdetermined.

The compounds AB_(—)0001251 through AB_(—)0001259 and AB_(—)0003130through AB_(—)0003150, shown Table 2, where screened at the two higherantibody concentrations. The result showed that all compounds exceptAB_(—)0001251 were interacting with the antibody. It was also shown thatcompound AB_(—)0001255, which is the original compound AB_(—)0001250with an extension, has the strongest binding of these compounds in theassay. Further, the results also showed that substituents on thearomatic ring are indispensable for binding in this type of compoundssince the only negative result was obtained with the unsubstitutedvariants AB_(—)0001251 and AB_(—)0003150.

EXAMPLE 5 General Method for Attaching Ligand to Support

Sepharose™ HP (Amersham Biosciences, Uppsala, Sweden) that hadpreviously been derivatised with allyl glycidyl ether was activated withbromine and coupled with hexametylene-diamine according to a standardprotocol. The free amine content was determined to 17 μmol/mL gelaccording to a standard protocol.

2 mL of this gel was transferred to a reaction vessel together with 2 mLof DMF. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(0.15 mmol) and diisopropyl amine (0.1 mmol) was added and thesuspension was put on a shaker at 30° C. After 5 min. the ligand to becoupled (0.1 mmol) was added and the reaction was allowed to continuefor 15 hours.

Thereafter the gel was transferred to a glass filter funnel and washedwith a 1:1 mixture of DMF and acetic acid anhydride. The gel was allowedto be in contact with this solution for 30 min. whereafter it was washedwith consecutively DMF, water, and 20% ethanol.

The amount of ligand coupled to the gel was determined with a NMR-methodusing tri-methoxy benzene as internal reference.

EXAMPLE 6 Chromatographic Characterisation of Affinity Media Containingthe Ligand AB 0003291 According to the Invention

AB_(—)0003291 coupled to Sepharose™ HP (ligand concentration 11 μmol/mlgel as determined by MAS-NMR) was packed in 0.5 ml Tricorn™ 5/20 columns(Amersham Biosciences, Uppsala, Sweden) at a flow rate of 1-2 ml/min.Monoclonals ABFab-K1 (FAB/kappa), ABFab-K2 (Fab′2/kappa) and ABFab-L2(Fab/lambda) were tested for binding to AB_(—)0003291-containing gelusing protein concentrations of 0.4 or 0.2 mg/ml in PBS, 1 M (NH₄)₂SO₄,pH 7. 100 μg of ABFab-K1 and 50 μg of the other two proteins wereinjected at a flow rate of 0.25 ml/min (contact time 2 min) using anÄkta™ Explorer 10 chromatography system equipped with a UV cell, pHmeter, conductivity cell and auto-injector (Amersham Biosciences,Uppsala, Sweden). Protein loading was followed by a wash period of 20column volumes of loading buffer (PBS, 1 M (NH₄)₂SO₄, pH 7) and a secondwash step with 0.01 M of NaOH.

FIGS. 7, 8 and 9 show the separate injections of the monoclonalsABFab-K1, ABFab-K2 and ABFab-L2, respectively, on theAB_(—)0003291-containing media. Evidently, the affinity columncontaining AB_(—)0003290 coupled to Sepharose™ HP is able to bind themonoclonals ABFab-K1 and ABFab-K2, whereas monoclonal ABFab-L2 directlycomes off the affinity column and is found in the flow-through. Elutionof monoclonals ABFab-K1 and ABFab-K2 bound to the affinity column inPBS, 1 M (NH₄)₂SO₄, pH 7 is possible using different elution conditionssuch as 50 mM acetate buffer containing 0.14 M NaCl, pH 4 or PBS, pH 7containing 10% n-propanol (data not shown).

1. A compound comprising formula (I)

wherein R₁ is CH₃ or CH₂CH₃; R₂ is a para and/or meta substituted phenyl group; R₃ is H, CH₃ or CH₂CH₃; and R₄ is a linear or cyclic aliphatic group, or, wherein R₁ and R₂ are as stated above while R₃ and R₄ are parts of a 4- to 6-membered cyclic entity, and which compound has affinity for human IgG of κ-type.
 2. The compound of claim 1, which is an affinity ligand with affinity for the constant region of a Fab fragment of human IgG of κ-type.
 3. The compound of claim 1, wherein R₁ is CH₃.
 4. The compound of claim 1, wherein R₂ is a substituted phenyl group having substituents selected from the group consisting of F, Cl, Br, I and O.
 5. The compound of claim 1, wherein the phenyl group of R₂ is substituted in the para position with a group —O—R₅, wherein R₅ is either CH₃ or CH₂CH₃.
 6. The compound of claim 4, wherein the phenyl group of R₂ is substituted with Cl or F in the meta position.
 7. The compound of claim 4, wherein the phenyl group of R₂ is substituted with Cl in meta and para position.
 8. The compound of claim 1, wherein R₄ is an aliphatic group, which includes oxygen atoms in one or more positions.
 9. The compound of claim 1, wherein R₄ is an aliphatic group, which contains one or more carbonyl groups.
 10. The compound of claim 1, wherein R₄ is an aliphatic group which includes a terminating functionality selected from the group consisting of a carboxylic acid, nitrogen, oxygen, sulphur or any derivative thereof.
 11. The compound of claim 1, wherein R₁ is CH₃; R₂ is a phenyl group that has been substituted with Cl in meta and para position; and R₃ and R₄ are parts of a cyclic 5-membered group.
 12. The compound of claim 11, wherein the cyclic 5-membered entity is substituted in a position directly adjacent to N with a C(O)—O—CH3 group.
 13. The compound of claim 1, which is capable of binding to the constant region of a human IgG of K-type, or a functional derivative thereof, with a binding constant of at least 10⁻³ M.
 14. The compound of claim 1, which is capable of binding to the constant region of a human IgG of κ-type, or a functional derivative thereof, via a binding pocket defined by the structure coordinates of the amino acids as shown in FIG.
 6. 15. (canceled)
 16. A sorption complex comprising the compound of claim 1 directly linked to the constant region of a Fab fragment of a human IgG of κ-type, or a functional derivative thereof.
 17. A separation matrix for affinity chromatography, comprising ligands coupled to a support, wherein the majority of the ligands are the compounds of claim
 1. 18. The separation matrix of claim 17, wherein the ligands have been coupled to the support via linkers.
 19. The separation matrix of claim 17, wherein the support is a porous polymeric particle.
 20. (canceled)
 21. A system suitable for affinity chromatography, comprising the separation matrix of claim 17 packed in a column. 